Proinflammatory cytokines are thought to play a significant role in the pathogenesis of type 2 diabetes (T2D) and are elevated in the circulation even before the onset of the disease. However, the full complement of cytokines involved in the development of T2D is not known. In this study, 32 serum cytokines were measured from diabetes-prone BKS.Cg-m+/+Lepr(db)/J (db/db) mice and heterozygous age-matched control mice at 5 weeks (non-diabetic/non-obese), 6-7 weeks (transitional-to-diabetes), or 11 weeks (hyperglycemic/obese) and then correlated with body weight, blood glucose, and fat content. Among these 32 cytokines, C-X-C motif ligand 1 (CXCL1) showed the greatest increase (+78%) in serum levels between db/db mice that were hyperglycemic (blood glucose: 519±23 mg/dl, n=6) and those that were non-hyperglycemic (193±13 mg/dl, n=8). Similarly, increased CXCL1 (+68%) and CXCL5 (+40%) were associated with increased obesity in db/db mice; note that these effects could not be entirely separated from age. We then examined whether islets could be a source of these chemokines. Exposure to cytokines mimicking low-grade systemic inflammation (10 pg/ml IL1β+20 pg/ml IL6) for 48 h upregulated islet CXCL1 expression by 53±3-fold and CXCL5 expression by 83±10-fold (n=4, P<0.001). Finally, overnight treatment with the combination of CXCL1 and CXCL5 at serum levels was sufficient to produce a significant decrease in the peak calcium response to glucose stimulation, suggesting reduced islet function. Our findings demonstrated that CXCL1 and CXCL5 i) are increased in the circulation with the onset of T2D, ii) are produced by islets under stress, and iii) synergistically affect islet function, suggesting that these chemokines participate in the pathogenesis of T2D.

Blood-borne neutrophils are excluded from entering lymph nodes across vascular portals termed high endothelial venules (HEVs) because of lack of expression of the CCR7 homeostatic chemokine receptor. Induction of sterile inflammation increases neutrophil entry into tumor-draining lymph nodes (TDLNs), which is critical for induction of antitumor adaptive immunity following treatments such as photodynamic therapy (PDT). However, the mechanisms controlling neutrophil entry into TDLNs remain unclear. Prior evidence that IL-17 promotes neutrophil emigration to sites of infection via induction of CXCL2 and CXCL1 inflammatory chemokines raised the question of whether IL-17 contributes to chemokine-dependent trafficking in TDLNs. In this article, we demonstrate rapid accumulation of IL-17-producing Th17 cells in the TDLNs following induction of sterile inflammation by PDT. We further report that nonhematopoietic expression of IL-17RA regulates neutrophil accumulation in TDLNs following induction of sterile inflammation by PDT. We show that HEVs are the major route of entry of blood-borne neutrophils into TDLNs through interactions of l-selectin with HEV-expressed peripheral lymph node addressin and by preferential interactions between CXCR2 and CXCL2 but not CXCL1. CXCL2 induction in TDLNs was mapped in a linear pathway downstream of IL-17RA-dependent induction of IL-1β. These results define a novel IL-17-dependent mechanism promoting neutrophil delivery across HEVs in TDLNs during acute inflammatory responses.

Patients with acute kidney injury (AKI) have increased serum proinflammatory cytokines and an increased occurrence of respiratory complications. The aim of the present study was to examine the effect of renal and extrarenal cytokine production on AKI-mediated lung injury in mice. C57Bl/6 mice underwent sham surgery, splenectomy, ischemic AKI, or ischemic AKI with splenectomy and kidney, spleen, and liver cytokine mRNA, serum cytokines, and lung injury were examined. The proinflammatory cytokines IL-6, CXCL1, IL-1β, and TNF-α were increased in the kidney, spleen, and liver within 6 h of ischemic AKI. Since splenic proinflammatory cytokines were increased, we hypothesized that splenectomy would protect against AKI-mediated lung injury. On the contrary, splenectomy with AKI resulted in increased serum IL-6 and worse lung injury as judged by increased lung capillary leak, higher lung myeloperoxidase activity, and higher lung CXCL1 vs. AKI alone. Splenectomy itself was not associated with increased serum IL-6 or lung injury vs. sham. To investigate the mechanism of the increased proinflammatory response, splenic production of the anti-inflammatory cytokine IL-10 was determined and was markedly upregulated. To confirm that splenic IL-10 downregulates the proinflammatory response of AKI, IL-10 was administered to splenectomized mice with AKI, which reduced serum IL-6 and improved lung injury. Our data demonstrate that AKI in the absence of a counter anti-inflammatory response by splenic IL-10 production results in an exuberant proinflammatory response and lung injury.

We showed previously that wild-type mice primed via intranasal inoculation with live or heat-inactivated Lactobacillus species were fully (100%) protected against the lethal sequelae of infection with the virulent pathogen, pneumonia virus of mice (PVM), a response that is associated with diminished expression of proinflammatory cytokines and diminished virus recovery. We show here that 40% of the mice primed with live Lactobacillus survived when PVM challenge was delayed for 5months. This robust and sustained resistance to PVM infection resulting from prior interaction with an otherwise unrelated microbe is a profound example of heterologous immunity. We undertook the present study in order to understand the nature and unique features of this response. We found that intranasal inoculation with L. reuteri elicited rapid, transient neutrophil recruitment in association with proinflammatory mediators (<em>CXCL1</em>, CCL3, CCL2, <em>CXCL1</em>0, TNF-alpha and IL-17A) but not Th1 cytokines. IFNγ does not contribute to survival promoted by Lactobacillus-priming. Live L. reuteri detected in lung tissue underwent rapid clearance, and was undetectable at 24h after inoculation. In contrast, L. reuteri peptidoglycan (PGN) and L. reuteri genomic DNA (gDNA) were detected at 24 and 48h after inoculation, respectively. In contrast to live bacteria, intranasal inoculation with isolated L. reuteri gDNA elicited no neutrophil recruitment, had minimal impact on virus recovery and virus-associated production of CCL3, and provided no protection against the negative sequelae of virus infection. Isolated PGN elicited neutrophil recruitment and proinflammatory cytokines but did not promote sustained survival in response to subsequent PVM infection. Overall, further evaluation of the responses leading to Lactobacillus-mediated heterologous immunity may provide insight into novel antiviral preventive modalities.

BACKGROUND

Lung ischemia-reperfusion (IR) injury leads to significant morbidity and mortality which remains a major obstacle after lung transplantation. However, the role of various subset(s) of lung cell populations in the pathogenesis of lung IR injury and the mechanisms of cellular protection remain to be elucidated. In the present study, we investigated the effects of adenosine A2A receptor (A2AAR) activation on resident lung cells after IR injury using an isolated, buffer-perfused murine lung model.

METHODS

To assess the protective effects of A2AAR activation, three groups of C57BL/6J mice were studied: a sham group (perfused for 2 hr with no ischemia), an IR group (1 hr ischemia + 1 hr reperfusion) and an IR+ATL313 group where ATL313, a specific A2AAR agonist, was included in the reperfusion buffer after ischemia. Lung injury parameters and pulmonary function studies were also performed after IR injury in A2AAR knockout mice, with or without ATL313 pretreatment. Lung function was assessed using a buffer-perfused isolated lung system. Lung injury was measured by assessing lung edema, vascular permeability, cytokine/chemokine activation and myeloperoxidase levels in the bronchoalveolar fluid.

RESULTS

After IR, lungs from C57BL/6J wild-type mice displayed significant dysfunction (increased airway resistance, pulmonary artery pressure and decreased pulmonary compliance) and significant injury (increased vascular permeability and edema). Lung injury and dysfunction after IR were significantly attenuated by ATL313 treatment. Significant induction of TNF-alpha, KC (CXCL1), MIP-2 (CXCL2) and RANTES (CCL5) occurred after IR which was also attenuated by ATL313 treatment. Lungs from A2AAR knockout mice also displayed significant dysfunction, injury and cytokine/chemokine production after IR, but ATL313 had no effect in these mice.

CONCLUSIONS

Specific activation of A2AARs provides potent protection against lung IR injury via attenuation of inflammation. This protection occurs in the absence of circulating blood thereby indicating a protective role of A2AAR activation on resident lung cells such as alveolar macrophages. Specific A2AAR activation may be a promising therapeutic target for the prevention or treatment of pulmonary graft dysfunction in transplant patients.

OBJECTIVE

AMP-activated protein kinase (AMPK) is metabolic biosensor with anti-inflammatory activities. Gout is commonly associated with excesses in soluble urate and in nutrition, both of which suppress tissue AMPK activity. Gout is driven by macrophage-mediated inflammation transduced partly by NLRP3 inflammasome activation and interleukin (IL)-1β release. Hence, we tested the hypothesis that AMPK activation limits monosodium urate (MSU) crystal-induced inflammation.

METHODS

We studied bone marrow-derived macrophages (BMDMs) from AMPKα1 knockout and wild-type mice, and assessed the selective AMPK pharmacological activator A-769662 and a low concentration (10 nM) of colchicine. We examined phosphorylation (activation) of AMPKα Thr172, NLRP3 mRNA expression, and caspase-1 cleavage and IL-1β maturation using western blot and quantitative RT-PCR approaches. We also assessed subcutaneous murine air pouch inflammatory responses to MSU crystals in vivo.

RESULTS

MSU crystals suppressed phosphorylation of AMPKα in BMDMs. Knockout of AMPKα1 enhanced, and, conversely, A-769662-inhibited MSU crystal-induced inflammatory responses including IL-1β and CXCL1 release in vitro and in vivo. A-769662 promoted AMPK-dependent macrophage anti-inflammatory M2 polarisation and inhibited NLRP3 gene expression, activation of caspase-1 and IL-1β. Colchicine, at low concentration (10 nM) achieved in gout flare prophylaxis dosing, promoted phosphorylation of AMPKα and macrophage M2 polarisation, and reduced activation of caspase-1 and release of IL-1β and CXCL1 by MSU crystals in BMDMs in vitro.

CONCLUSIONS

AMPK activity limits MSU crystal inflammation in vitro and in vivo, and transduces multiple anti-inflammatory effects of colchicine in macrophages. Targeting increased and sustained AMPK activation in inflammatory cells merits further investigation for enhancing efficacy of prophylaxis and treatment of gouty inflammation.

The tumour microenvironment (TME) shapes disease progression and influences therapeutic response. Most aggressive solid tumours have high levels of myeloid cell infiltration, namely tumour associated macrophages (TAM). Recapitulation of the interaction between the different cellular players of the TME, along with the extracellular matrix (ECM), is critical for understanding the mechanisms underlying disease progression. This particularly holds true for prediction of therapeutic response(s) to standard therapies and interrogation of efficacy of TME-targeting agents. In this work, we explored a culture platform based on alginate microencapsulation and stirred culture systems to develop the 3D-3-culture, which entails the co-culture of tumour cell spheroids of non-small cell lung carcinoma (NSCLC), cancer associated fibroblasts (CAF) and monocytes. We demonstrate that the 3D-3-culture recreates an invasive and immunosuppressive TME, with accumulation of cytokines/chemokines (IL4, IL10, IL13, CCL22, CCL24, CXCL1), ECM elements (collagen type I, IV and fibronectin) and matrix metalloproteinases (MMP1/9), supporting cell migration and promoting cell-cell interactions within the alginate microcapsules. Importantly, we show that both the monocytic cell line THP-1 and peripheral blood-derived monocytes infiltrate the tumour tissue and transpolarize into an M2-like macrophage phenotype expressing CD68, CD163 and CD206, resembling the TAM phenotype in NSCLC. The 3D-3-culture was challenged with chemo- and immunotherapeutic agents and the response to therapy was assessed in each cellular component. Specifically, the macrophage phenotype was modulated upon treatment with the CSF1R inhibitor BLZ945, resulting in a decrease of the M2-like macrophages. In conclusion, the crosstalk between the ECM and tumour, stromal and immune cells in microencapsulated 3D-3-culture promotes the activation of monocytes into TAM, mimicking aggressive tumour stages. The 3D-3-culture constitutes a novel tool to study tumour-immune interaction and macrophage plasticity in response to external stimuli, such as chemotherapeutic and immunomodulatory drugs.

Interleukin-10 (IL-10) modulates inflammatory responses elicited in vitro and in vivo by Borrelia burgdorferi, the Lyme disease spirochete. How IL-10 modulates these inflammatory responses still remains elusive. We hypothesize that IL-10 inhibits effector functions of multiple genes induced by B. burgdorferi in macrophages to control concomitantly elicited inflammation. Because macrophages are essential in the initiation of inflammation, we used mouse J774 macrophages and live B. burgdorferi spirochetes as the model target cell and stimulant, respectively. First, we employed transcriptome profiling to identify genes that were induced by stimulation of cells with live spirochetes and that were perturbed by addition of IL-10 to spirochete cultures. Spirochetes significantly induced upregulation of 347 genes at both the 4-h and 24-h time points. IL-10 inhibited the expression levels, respectively, of 53 and 65 of the 4-h and 24-h genes, and potentiated, respectively, at 4 h and 24 h, 65 and 50 genes. Prominent among the novel identified IL-10-inhibited genes also validated by quantitative real-time PCR (qRT-PCR) were Toll-like receptor 1 (TLR1), TLR2, IRAK3, TRAF1, IRG1, PTGS2, MMP9, IFI44, IFIT1, and CD40. Proteome analysis using a multiplex enzyme-linked immunosorbent assay (ELISA) revealed the IL-10 modulation/and or potentiation of RANTES/CCL5, macrophage inflammatory protein 2 (MIP-2)/CXCL2, IP-10/CXCL1CXCL1, CXCL5, CCL2, CCL4, IL-6, tumor necrosis factor alpha (TNF-α), IL-1α, IL-1β, gamma interferon (IFN-γ), and IL-9. Similar results were obtained using sonicated spirochetes or lipoprotein as stimulants. Our data show that IL-10 alters effectors induced by B. burgdorferi in macrophages to control concomitantly elicited inflammatory responses. Moreover, for the first time, this study provides global insight into potential mechanisms used by IL-10 to control Lyme disease inflammation.

Ribonucleic acid interference (RNAi) based on microRNA (miRNA) context may provide an efficient and safe therapeutic knockdown effect and can be driven by ribonucleic acid polymerase II (RNAP II). In this study, we designed and synthesized miR155-based artificial miRNAs against heparanase (HPSE) constructed with BLOCK-iT™ Pol II miR RNAi Expression Vector Kit. The expression levels of HPSE declined significantly in both the mRNA and protein levels in HPSE-miRNA transfected melanoma cells that exhibited reduction of adhesion, migration, and invasion ability in vitro and in vivo. We also observed that HPSE miRNA could inhibit the expressions of chemokines of interleukin-8 (IL8) and chemokine (C-X-C motif) ligand 1 (CXCL1), at both the transcriptional and translational levels. Further study on its probable mechanism declared that down-regulation of IL8 and CXCL1 by HPSE-miRNA may be correlated with reduced growth-factor simulated mitogen-activated kinase (MAPK) phosphorylation including p38 MAPK, c-Jun N-terminal kinase (JNK) and extracellular-signal-regulated kinase (ERK) 1 and 2, which could be rescued by miRNA incompatible mutated HPSE cDNA. In conclusion, we demonstrated that artificial miRNAs against HPSE might serve as an alterative mean of therapy to low HPSE expression and to block the adhesion, invasion, and metastasis of melanoma cells. Furthermore, miRNA-based RNAi was also a powerful tool for gene function study.

BACKGROUND

Mucosal CXC chemokines recruit inflammatory cells to the infected urinary tract. The chemokine response repertoire of the urinary tract and the relationship to disease severity have not been examined, however.

METHODS

This study quantified CXC (<em>CXCL1</em>, CXCL3, CXCL5, CXCL8, CXCL9, and <em>CXCL1</em>0) and CC (CCL2, CCL4, and CCL5) chemokines in sequential urine samples obtained from 50 patients with febrile urinary tract infections during 24 hours after diagnosis.

RESULTS

All patients had elevated chemokine levels, but bacteremic infections caused higher <em>CXCL1</em>, CXCL3, CXCL5, CXCL8, and CCL2 responses. CCL2 and CXCL8 levels were higher in patients with acute pyelonephritis symptoms and CCL2, CXCL3, CCL4, CXCL5, and <em>CXCL1</em>0 were significantly correlated to C-reactive protein (CRP) and temperature. Women and men showed different chemokine responses.

CONCLUSIONS

Febrile urinary tract infections are accompanied by a complex chemokine response. The response magnitude reflects disease severity, and the repertoire is influenced by gender and underlying disease.

The chemokine CXCL1 and its receptor CXCR2 play a crucial role in host immune response by recruiting and activating neutrophils for microbial killing at the tissue site. Dysregulation in this process has been implicated in collateral tissue damage causing disease. CXCL1 reversibly exists as monomers and dimers, and it has been proposed that distinct monomer and dimer activities and the monomer-dimer equilibrium regulate the neutrophil function. However, the molecular mechanisms linking the CXCL1/CXCR2 axis and the neutrophil 'beneficial' and 'destructive' phenotypes are not known. In this study, we characterized neutrophil trafficking and its consequence in the mouse lung by the CXCL1 wild type (WT), which exists as monomers and dimers, and by a nondissociating dimer. Whereas the WT, compared to the dimer, was more active at low doses, both the WT and the dimer elicited a large neutrophil efflux at high doses. Importantly, robust neutrophil recruitment elicited by the WT or dimer was not detrimental to lung tissue integrity and, further, could not be correlated to surface CXCR2 levels. We conclude that the CXCL1 monomer-dimer distribution and receptor interactions are highly coupled and regulate neutrophil trafficking and that injury in the context of disease is a consequence of inappropriate CXCR2 activation at the target tissue and not due to mechanical forces exerted by neutrophils during recruitment.

BACKGROUND

Toll-Like receptors (TLRs) belong to the family of pattern-recognition receptors with a crucial function of recognising pathogen-associated molecular patterns (PAMPs). Cryptococcal meningitis is a potentially fatal disease with a high mortality and risk of neurological sequelae.

METHODS

We studied the ability of microglial cells to increase the phagocytosis of cryptococci after stimulation with agonists of TLR1/2, TLR3, TLR4 and TLR9.

RESULTS

Stimulation of murine microglial cells with these TLR agonists for 24 h increased the phagocytosis of encapsulated Cryptococcus neoformans. Stimulation increased the release of TNF-α, CXCL1 (KC), IL-6, IL-10 and MIP-2, which indicated the activation of microglial cells. Unstimulated and TLR agonist-stimulated MyD88-deficient cells showed a reduced ability to phagocytose cryptococci compared to their wild-type counterpart. Intracellular killing of cryptococci was also increased in TLR-stimulated cells compared to unstimulated microglial cells.

CONCLUSIONS

Our observation suggests that stimulation of microglial cells by TLR agonists can increase the resistance of the brain against CNS infections caused by Cryptococcus neoformans. This may be of interest when an immunocompromised patient is unable to eliminate Cryptococcus neoformans despite antifungal therapy.

Citrobacter rodentium infection of mice induces cell-mediated immune responses associated with crypt hyperplasia and epithelial β-catenin signaling. Recent data suggest that phosphatidylinositol-3-kinase (PI3K)/Akt signaling cooperates with Wnt to activate β-catenin in intestinal stem and progenitor cells through phosphorylation at Ser552 (P-β-catenin(552)). Our aim was to determine whether epithelial PI3K/Akt activation is required for β-catenin signaling and host defense against C. rodentium. C57BL/6 mice were infected with C. rodentium and treated with dimethyl sulfoxide (DMSO) (vehicle control) or with the PI3K inhibitor LY294002 or wortmannin. The effects of infection on PI3K activation and β-catenin signaling were analyzed by immunohistochemistry. The effects of PI3K inhibition on host defense were analyzed by the quantification of splenic and colon bacterial clearance, and adaptive immune responses were measured by real-time PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Increased numbers of P-β-catenin(552)-stained epithelial cells were found throughout expanded crypts in C. rodentium colitis. We show that the inhibition of PI3K signaling attenuates epithelial Akt activation, the Ser552 phosphorylation and activation of β-catenin, and epithelial cell proliferative responses during C. rodentium infection. PI3K inhibition impairs bacterial clearance despite having no impact on mucosal cytokine (gamma interferon [IFN-γ], tumor necrosis factor [TNF], interleukin-17 [IL-17], and IL-1β) or chemokine (<em>CXCL1</em>, CXCL5, CXCL9, and <em>CXCL1</em>0) induction. The results suggest that the host defense against C. rodentium requires epithelial PI3K activation to induce Akt-mediated β-catenin signaling and the clearance of C. rodentium independent of adaptive immune responses.

Chronic obstructive pulmonary disease is mainly triggered by cigarette smoke (CS) and progresses even after smoking cessation. CS induces an exaggerated influx of inflammatory cells to the bronchoalveolar space and lung parenchyma, likely resulting from a complex interplay between chemoattractants and their respective receptors. In a murine CS model of chronic obstructive pulmonary disease, we studied the importance of chemokine-like receptor ChemR23 for the induction and resolution of inflammation in CS-exposed lungs. Subacute and chronic CS exposure increased protein levels of the ChemR23 ligand and chemoattractant, chemerin, in bronchoalveolar lavage (BAL) fluid of wild-type (WT) mice. Moreover, the proinflammatory chemokines CXCL1, CCL2, and CCL20 were increased in the airways of CS-exposed WT mice, accompanied by a massive accumulation of inflammatory neutrophils and monocytes, CD11b(hi)CD103(-) and CD11b(lo)CD103(+) dendritic cells (DCs), and CD4(+) and CD8(+) T cells. The lung parenchyma of WT mice was infiltrated with inflammatory neutrophils, CD11b(hi)CD103(-) DCs, and activated CD4(+) T cells after CS exposure. CS-induced inflammation was severely attenuated in BAL fluid and lungs of ChemR23 knockout mice with regard to the induction of inflammatory chemokines and the recruitment of inflammatory cells. Neutrophils and CD8(+) T cells persisted in the airways of WT mice, as did the airway-derived conventional DCs in the mediastinal lymph nodes, for at least 14 d after smoking cessation. In the BAL fluid of CS-exposed ChemR23 knockout mice, there was a remarkable delayed accumulation of T cells 14 d after the final exposure. Our data support a role for ChemR23 in directing innate and adaptive immune cells to CS-exposed lungs.

To assess early changes in the lung after low-dose radiation exposure that may serve as targets for mitigation of lung injury in the aftermath of a terrorist event, we analyzed cytokine expression after irradiation. Adult mice were studied after whole-lung or total-body irradiation. Mouse pups of different ages were also investigated after total-body irradiation. mRNA abundance was analyzed in tissue and plasma, and pathological changes were assessed. In lung tissue, dose-related changes were seen in IL1B, IL1R2 and CXCR2 mRNA expression at 1 and 6 h after irradiation, concurrent with increases in plasma protein levels of KC/CXCL1 and IL6. However, in the pups, changes in IL1 abundance were not detected until 28 days of age, coincident with the end of postnatal lung growth, although apoptosis was detected at all ages. In conclusion, although cytokines were expressed after low doses of radiation, their role in the progression of tissue response is yet to be determined. They may be candidates for use in marker-based biodosimetry. However, the lack of cytokine induction in early life suggests that different end points (and mitigating treatments) may be required for children.

In neutrophils, growth-related protein-alpha (CXCL1) and interleukin-8 (CXCL8), are potent chemoattractants (Cytokine 14:27-36, 2001; Biochemistry 42:2874-2886, 2003) and can stimulate myeloperoxidase release via activation of the G protein-coupled receptors CXCR1 and CXCR2. The role of CXCR1 and CXCR2 in the pathogenesis of inflammatory responses has encouraged the development of small molecule antagonists for these receptors. The data presented herein describe the pharmacology of 2-hydroxy-N,N-dimethyl-3-{2-[[(R)-1-(5-methyl-furan-2-yl)-propyl]amino]-3,4-dioxo-cyclobut-1-enylamino}-benzamide (Sch527123), a novel antagonist of both CXCR1 and CXCR2. Sch527123 inhibited chemokine binding to (and activation of) these receptors in an insurmountable manner and, as such, is categorized as an allosteric antagonist. Sch527123 inhibited neutrophil chemotaxis and myeloperoxidase release in response to CXCL1 and CXCL8 but had no effect on the response of these cells to C5a or formyl-methionyl-leucyl-phenylalanine. The pharmacological specificity of Sch527123 was confirmed by testing in a diversity profile against a panel of enzymes, channels, and receptors. To measure compound affinity, we characterized [(3)H]Sch527123 in both equilibrium and nonequilibrium binding analyses. Sch527123 binding to CXCR1 and CXCR2 was both saturable and reversible. Although Sch527123 bound to CXCR1 with good affinity (K(d) = 3.9 +/- 0.3 nM), the compound is CXCR2-selective (K(d) = 0.049 +/- 0.004 nM). Taken together, our data show that Sch527123 represents a novel, potent, and specific CXCR2 antagonist with potential therapeutic utility in a variety of inflammatory conditions.

Pro-inflammatory mediators, like prostaglandin (PG) and chemokines, promote tumourigenesis by enhancing cell proliferation, migration of immune cells and recruitment of blood vessels. Recently we showed elevated expression of the chemokine (C-X-C motif) receptor 2 (CXCR2) in endometrial adenocarcinomas localized to neutrophils and neoplastic epithelial and vascular cells. Furthermore we found that PGF(2alpha)-F-prostanoid (FP) receptor regulates the expression of the CXCR2 ligand CXCL1, to promote neutrophil chemotaxis in endometrial adenocarcinomas. In the present study we identified another CXCR2 ligand, CXCL8 as a target for PGF(2alpha)-FP receptor signalling which enhances epithelial cell proliferation in endometrial adenocarcinoma cells in vitro and in nude mice in vivo. We found that PGF(2alpha)-FP receptor interaction induces CXCL8 expression in endometrial adenocarcinoma cells via the protein kinase C-calcium-calcineurin-NFAT signaling pathway. Promoter analysis revealed that CXCL8 transcriptional activation by PGF(2alpha) signaling is mediated by cooperative interactions between the AP1 and NFAT binding sites. Furthermore, PGF(2alpha) via the FP receptor induced the expression of the regulator of calcineurin 1 isoform 4 (RCAN1-4) via the calcineurin/NFAT pathway in a reciprocal manner to CXCL8. Using an adenovirus to overexpress RCAN1-4, we found that RCAN1-4 is a negative regulator of CXCL8 expression in endometrial adenocarcinoma cells. Taken together our data have elucidated the molecular and cellular mechanism whereby PGF(2alpha) regulates CXCL8 expression via the FP receptor in endometrial adenocarcinomas and have highlighted RCAN1-4 as a negative regulator of CXCL8 expression which may be exploited therapeutically to inhibit CXCL8-mediated tumour development.

Previous studies have indicated that group B streptococcus (GBS), a frequent human pathogen, potently induces the release of interleukin-1β (IL-1β), an important mediator of inflammatory responses. Since little is known about the role of this cytokine in GBS disease, we analyzed the outcome of infection in IL-1β-deficient mice. These animals were markedly sensitive to GBS infection, with most of them dying under challenge conditions that caused no deaths in wild-type control mice. Lethality was due to the inability of the IL-1β-deficient mice to control local GBS replication and dissemination to target organs, such as the brain and the kidneys. Moreover, in a model of inflammation induced by the intraperitoneal injection of killed GBS, a lack of IL-1β was associated with selective impairment in the production of the neutrophil chemokines CXCL1 and CXCL2 and in neutrophil recruitment to the peritoneal cavity. Decreased blood neutrophil counts and impaired neutrophil recruitment to the brain and kidneys were also observed during GBS infection in IL-1β-deficient mice concomitantly with a reduction in CXCL1 and CXCL2 tissue levels. Notably, the hypersusceptibility to GBS infection observed in the immune-deficient animals was recapitulated by neutrophil depletion with anti-Gr1 antibodies. Collectively, our data identify a cytokine circuit that involves IL-1β-induced production of CXCL1 and CXCL2 and leads the recruitment of neutrophils to GBS infection sites. Moreover, our data point to an essential role of these cells in controlling the progression and outcome of GBS disease.

Macrophage migration inhibitory factor (MIF) has been defined as an important chemokine-like function (CLF) chemokine with an essential role in monocyte recruitment and arrest. Adhesion of monocytes to the vessel wall and their transendothelial migration are critical in atherogenesis and many other inflammatory diseases. Chemokines carefully control all steps of the monocyte recruitment process. Those chemokines specialized in controlling arrest are typically immobilized on the endothelial surface, mediating the arrest of rolling monocytes by chemokine receptor-triggered pathways. The chemokine receptor CXCR2 functions as an important arrest receptor on monocytes. An arrest function has been revealed for the bona fide CXCR2 ligands <em>CXCL1</em> and CXCL8, but genetic studies also suggested that additional arrest chemokines are likely to be involved in atherogenic leukocyte recruitment. While CXCR2 is known to interact with numerous CXC chemokine ligands, the CLF chemokine MIF, which structurally does not belong to the CXC chemokine sub-family, was surprisingly identified as a non-cognate ligand of CXCR2, responsible for critical arrest functions during the atherogenic process. MIF was originally identified as macrophage migration inhibitory factor (this function being eponymous), but is now known as a potent inflammatory cytokine with CLFs including chemotaxis and leukocyte arrest. This review will cover the mechanisms underlying these functions, including MIF's effects on LFA1 integrin activity and signal transduction, and will discuss the structural similarities between MIF and the bona fide CXCR2 ligand CXCL8 while emphasizing the structural differences. As MIF also interacts with CXCR4, a chemokine receptor implicated in <em>CXCL1</em>2-elicited lymphocyte arrest, the arrest potential of the MIF/CXCR4 axis will also be scrutinized as well as the recently identified role of pericyte MIF in attracting leukocytes exiting through venules as part of the pericyte "motility instruction program."

Ozone is known to produce an acute influx of neutrophils, and alveolar epithelial cells can secrete chemokines and modulate inflammatory processes. However, direct exposure of alveolar epithelial cells and macrophages to ozone (O(3)) produces little chemokine response. To determine if cell-cell interactions might be responsible, we investigated the effect of alveolar macrophage-conditioned media after ozone exposure (MO(3)CM) on alveolar epithelial cell chemokine production. Serum-free media were conditioned by exposing a rat alveolar macrophage cell line NR8383 to ozone for 1 hour. Ozone stimulated secretion of IL-1alpha, IL-1beta, and IL-18 from NR8383 cells, but there was no secretion of chemokines or TNF-alpha. Freshly isolated type II cells were cultured, so as to express the biological markers of type I cells, and these cells are referred to as type I-like cells. Type I-like cells were exposed to diluted MO(3)CM for 24 hours, and this conditioned medium stimulated secretion of cytokine-induced neutrophil chemattractant-1 (CXCL1) and monocyte chemoattractant protein-1 (CCL2). Secretion of these chemokines was inhibited by the IL-1 receptor antagonist. Although both recombinant IL-1alpha and IL-1beta stimulated alveolar epithelial cells to secrete chemokines, recombinant IL-1alpha was 100-fold more potent than IL-1beta. Furthermore, neutralizing anti-rat IL-1alpha antibodies inhibited the secretion of chemokines by alveolar epithelial cells, whereas neutralizing anti-rat IL-1beta antibodies had no effect. These observations indicate that secretion of IL-1alpha from macrophages stimulates alveolar epithelial cells to secrete chemokines that can elicit an inflammatory response.

Type I IFNs are used for treating viral, neoplastic, and inflammatory disorders. The protein products encoded by IFN-stimulated genes (ISGs) likely mediate clinical effects of IFN in patients. Macroarray assays, used for studying ISG induction in IFN-treated patients, comprise genes identified predominantly through analysis of long-term cell lines. To discover genes induced selectively by IFN-beta in PBMC, we exposed whole blood to physiological concentrations of IFN-beta. PBMC were prepared, and RNA was extracted, reverse-transcribed, and hybridized to cDNA microarrays, and microarray analysis identified 39 ISGs and 20 IFN-repressed genes (IRGs). Thirty-three ISGs were known previously, and six ISGs were novel. New ISGs included GTP cyclohydrolase 1; hypothetical protein LOC129607; hypothetical protein FLJ38348; leucine aminopeptidase 3; squalene epoxidase; and GTP-binding protein overexpressed in skeletal muscle. Twenty IRGs included IL-1beta and CXCL8, which had been identified earlier. CXCL1 was a novel IRG identified in the current study. PCR analysis demonstrated the regulation of six novel ISGs and CXCL1 as an IRG in PBMC and astrocytoma cells. Results were validated using RNA obtained ex vivo from blood of patients after injection with IFN-beta. Identification of new ISGs and IRGs in primary PBMC will enhance macroarray assays for monitoring IFN responsiveness.

Ceramide is recognized as an antiproliferative and proapoptotic sphingolipid metabolite; however, the role of ceramide in inflammation is not well understood. To determine the role of C6-ceramide in regulating inflammatory responses, human corneal epithelial cells were treated with C6-ceramide in 80 nm diameter nanoliposome bilayer formulation (Lip-C6) prior to stimulation with UV-killed Staphylococcus aureus. Lip-C6 (5 muM) inhibited the phosphorylation of proinflammatory and proapoptotic MAP kinases JNK and p38 and production of neutrophil chemotactic cytokines CXCL1, CXCL5, and CXCL8. Lip-C6 also blocked CXC chemokine production by human and murine neutrophils. To determine the effect of Lip-C6 in vivo, a murine model of corneal inflammation was used in which LPS or S. aureus added to the abraded corneal surface induces neutrophil infiltration to the corneal stroma, resulting in increased corneal haze. Mice were treated topically with 2 nMoles (811 ng) Lip-C6 or with control liposomes prior to, or following, LPS or S. aureus stimulation. We found that corneal inflammation was significantly inhibited by Lip-C6 but not control liposomes given prior to, or following, activation by LPS or S. aureus. Furthermore, Lip-C6 did not induce apoptosis of corneal epithelial cells in vitro or in vivo, nor did it inhibit corneal wound healing. Together, these findings demonstrate a novel, anti-inflammatory, nontoxic, therapeutic role for liposomally delivered short-chain ceramide.

Lymph node metastasis is a significant factor in gastric cancer prognosis. It is well known that cancer cells secrete lymphangiogenic factors, thereby promoting lymphangiogenesis. However, the effects of lymphatic endothelial cell (LEC)-secreted factors on the process of lymphangiogenesis and tumor cell metastasis remain unclear. We established an animal model and successfully isolated LECs from afferent lymph vessels of sentinel lymph nodes (SLNs) in animal models. A microarray analysis was performed to characterize gene expression profile in afferent LECs induced by metastatic cancer cells. There were significant differences in 846 genes between normal LECs and LECs with lymph node metastasis. Among these genes, we found that expression of CXCL1 was upregulated, which was confirmed by quantitative reverse-transcriptase polymerase chain reaction. In a coculture system, gastric cancer cells induced CXCL1 secretion from LECs, which was associated with the NF-κB pathway. CXCL1 stimulated LECs migration and tube formation involving FAK-ERK1/2-RhoA activation and reorganization of F-actin. In human gastric cancer specimens, CXCR2 expression was positively correlated with TNM (Tumor, node, metastasis) stage and lymphatic vessel density. These results suggested that LECs of afferent SLNs had specific expression profiles, which were distinct from those of normal lymphatic vessels and appeared to promote metastasis. The expression pattern described in our study, including CXCL1 in LECs and its receptor CXCR2 in cancer cells, offers a promising therapeutic target for gastric cancer.

Leptin, a pleiotropic type I cytokine, was recently demonstrated to be expressed by resident lung cells in chronic obstructive pulmonary disease patients and asymptomatic smokers. To elucidate the functional role of leptin in the onset of chronic obstructive pulmonary disease, we tested leptin-deficient ob/ob mice (C57BL/6), leptin receptor-deficient db/db mice (C57BKS), and littermates in a model of cigarette smoke (CS)-induced pulmonary inflammation. Wild-type (WT) C57BL/6 mice were exposed for 4 or 24 wk to control air or CS. Pulmonary leptin expression was analyzed by immunohistochemistry and real-time PCR. Pulmonary inflammation upon 4 wk CS exposure was evaluated in bronchoalveolar lavage fluid (BALF) and lung tissue of WT, ob/ob, and db/db mice. Immunohistochemical analysis revealed leptin expression in bronchial epithelial cells, pneumocytes, alveolar macrophages, and bronchial/vascular smooth muscle cells. The 4 and 24 wk CS exposure increased leptin expression in bronchial epithelial cells and pneumocytes versus air-exposed WT mice (p<0.05). The 4 wk CS exposure resulted in increased accumulation of neutrophils, dendritic cells, macrophages, and lymphocytes in BALF and lung tissue of WT, ob/ob, and db/db mice. CS-exposed ob/ob and db/db mice showed in general higher numbers of neutrophils and lower numbers of CD4+, CD8+, and dendritic cells versus CS-exposed WT mice. Consistently, CXCL1 levels were enhanced in BALF of CS-exposed ob/ob and db/db mice versus WT mice (p<0.05). Exogenous leptin administration completely restored the skewed inflammatory profile in ob/ob mice. These data reveal an important role of leptin in modulating innate and adaptive immunity after CS inhalation in mice.